For the past several years, my lab has made key discoveries of the paracellular channel underlying renal calcium and magnesium handling. At the center of our story is a key tight junction protein complex from the kidney that comprises of three claudin proteins: claudin-14, claudin-16 and claudin-19. Human mutations in claudin-16 and claudin-19 cause the hereditary disease FHHNC (Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis). Human SNPs in claudin-14 are associated with kidney stone diseases according to genome-wide association criteria. This proposal entitled Biochemical and biophysical nature of claudin-16 and claudin-19 channels will test the central hypothesis that the changes in paracellular permeabilities through claudin expression, function, interaction and combination underlie the key signaling functions of CaSR and PTH to regulate renal calcium and magnesium reabsorption. There are three aims in this proposal. Aim 1 will elucidate the biochemical and biophysical basis of claudin-14, -16 and -19 functions in the kidney. We will use a range of biochemical and biophysical approaches with emphasis on a novel recording approach established by our lab based upon Scanning Ion Conductance Microscopy (SICM). SICM has, for the first time, allowed us to reveal paracellular permeabilities at submicron resolution. Aim 2 will elucidate the regulatory basis for claudin in response to CaSR and PTH signaling. We will generate two key mouse models - TALH specific CaSR or PTH1R KO mice to decipher the CaSR and PTH specific regulation of claudins. We will also study how CaSR and PTH signaling cross talk in the kidney using double CaSR/PTH1R KO or a well-established pharmacological approach. Aim 3 will determine the mechanistic role of claudin in renal calcium handling. The renal transport function of claudin monomer or oligomer will be determined using combinatorial knockout approaches based upon the claudin KO mouse models we have established - claudin-14, -16, -19 single, double and triple KO and claudin- 14 overexpression mice. We will also study how PTH, CaSR and microRNA alter paracellular permeabilities based upon changes in claudin expression, function, interaction, and combination using single nephron perfusion techniques.